Synthesis and anticonvulsant activity of phenoxyacetyl derivatives of amines, including aminoalkanols and amino acids

Article abstract of DOI:10.1039/c8md00430g

A series of 17 new phenoxyacetamides has been prepared via multistep chemical synthesis as a continuation of the research carried out by our group on di- and tri-substituted phenoxyalkyl and phenoxyacetyl derivatives of amines. The obtained compounds vary in an amide component, for example aminoalkanol or (un)modified amino acid moieties were introduced. The structures of selected products were confirmed by means of crystallographic methods. All 17 compounds were the subject of preliminary screening for potential anticonvulsant activity (MES, 6 Hz and/or scMET tests) and neurotoxicity (rotarod) in mice after intraperitoneal administration, while several active compounds were subsequently examined in additional models (e.g. MES and rotarod-rats, p.o. or i.p., hippocampal kindling-rats, i.p.). Finally, safety studies (cytotoxicity and cell proliferation assays on astrocytes, metabolic stability assessment, mutagenicity evaluation) were performed for several active compounds, including the most promising one (R-(?)-2-(2,6-dimethylphenoxy)-N-(1-hydroxypropan-2-yl)acetamide, MES ED₅₀ = 12.00 mg per kg b.w., rats, p.o.).

Full text of DOI:10.1039/c8md00430g

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RESEARCH ARTICLE
Synthesis and anticonvulsant activity of
phenoxyacetyl derivatives of amines, including
aminoalkanols and amino acids†
Cite this: Med. Chem. Commun.,
2018, 9, 1933
a
*
Katarzyna Pańczyk,
Dorota Żelaszczyk, a Paulina Koczurkiewicz,b
Karolina Słoczyńska,b Elżbieta Pękala,b Ewa Żesławska,
Wojciech Nitek,d
c
Paweł Żmudzki,e Henryk Maronaa and Anna Waszkielewicz
a
A series of 17 new phenoxyacetamides has been prepared via multistep chemical synthesis as a continua-
tion of the research carried out by our group on di- and tri-substituted phenoxyalkyl and phenoxyacetyl
derivatives of amines. The obtained compounds vary in an amide component, for example aminoalkanol
or (un)modified amino acid moieties were introduced. The structures of selected products were con-
firmed by means of crystallographic methods. All 17 compounds were the subject of preliminary screen-
ing for potential anticonvulsant activity (MES, 6 Hz and/or scMET tests) and neurotoxicity (rotarod) in
mice after intraperitoneal administration, while several active compounds were subsequently examined
in additional models (e.g. MES and rotarod – rats, p.o. or i.p., hippocampal kindling – rats, i.p.). Finally,
safety studies (cytotoxicity and cell proliferation assays on astrocytes, metabolic stability assessment,
mutagenicity evaluation) were performed for several active compounds, including the most promising
one (R-(−)-2-(2,6-dimethylphenoxy)-N-(1-hydroxypropan-2-yl)acetamide, MES ED50 = 12.00 mg per kg
b.w., rats, p.o.).
Received 29th August 2018,
Accepted 20th September 2018
DOI: 10.1039/c8md00430g
rsc.li/medchemcomm
active phenoxyethyl derivatives di- and tri-substituted with
methyl groups in the phenyl ring and general conclusions de-
1. Introduction
Epilepsy is a neurological condition which affects approxi-
mately 65–70 million people worldwide.1 Despite the availabil-
ity of a huge number of anticonvulsants on the market, char-
acterized by various mechanisms of action, still about 40% of
patients do not respond to treatment.2 Such pharmaco-
resistance of epilepsy, as well as frequently observed adverse
effects,3 is a great premise for searching for new anticonvul-
sant compounds.
rived from our studies indicate that this type of structural
modification might be particularly favorable.4–7 Additionally,
SAR analysis showed that incorporation of 2-amino-propan-1-
ol as an amine moiety might be advantageous (e.g. reference
compound I, active in animal models of convulsions, show-
ing high affinity towards the receptors sigma, 5-HT1A, alpha-2
and 5-HT transporter, Fig. 1B).6 We also investigated some
phenoxyacetyl derivatives of aminoalkanols, as an amide
moiety is a scaffold known to be present in many anticonvul-
sants currently available on the market as well as compounds
under research (Fig. 1A). In the case of compounds disubsti-
tuted in the phenyl ring with chlorine and a methyl group
(in positions 2,5 and 4,2 or 4,3, respectively) the amides
lacked the desired activity.8,9 However, other types of substi-
tution seem to be promising, e.g. in the case of the 2,6-
dimethylphenoxyethyl derivative of 2-amino-1-cyclohexanol, in-
corporation of an amide group resulted in an active com-
pound (although slightly less potent than the amine analog)
(Fig. 1C, reference compound II).5
Within our previous research we investigated the anticon-
vulsant activity of many phenoxyalkyl and phenoxyethoxyethyl
derivatives of amino alcohols. Among them, there were many
a Department of Bioorganic Chemistry, Chair of Organic Chemistry, Faculty of
Pharmacy, Jagiellonian University Medical College, Medyczna 9, 30-688 Cracow,
Poland. E-mail: katarzyna.panczyk@uj.edu.pl
b Department of Pharmaceutical Biochemistry, Faculty of Pharmacy, Jagiellonian
University Medical College, Medyczna 9, 30-688 Cracow, Poland
c Department of Chemistry, Institute of Biology, Pedagogical University,
Podchorążych 2, 30-084 Cracow, Poland
d Faculty of Chemistry, Jagiellonian University, Gronostajowa 2, 30-387 Cracow,
Poland
e Department of Medicinal Chemistry, Faculty of Pharmacy, Jagiellonian
University Medical College, Medyczna 9, 30-688 Cracow, Poland
† Electronic supplementary information (ESI) available. CCDC 1832675–1832677.
For ESI and crystallographic data in CIF or other electronic format see DOI:
10.1039/c8md00430g
In the currently presented research, we decided to synthe-
size phenoxyacetyl derivatives which are di- or tri-substituted
in the phenyl ring with methyl groups and investigate their
anticonvulsant properties. The amine component of the
designed compounds varies in structure. Apart from amines
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Fig. 1 Chemical structures of anticonvulsants containing an amide moiety (A) and reference compounds I (B)6 and II (C).5
with/without an additional hydroxyl substituent (particularly
derivatives of 2-aminopropan-1-ol) that were already within
our interests, we decided to include an (un)modified amino
acid group in the structure of several compounds. Such mod-
ification resulted in compounds that may be the metabolic
products of aminoalkanol derivatives. Moreover, there are ex-
amples of the anticonvulsant activity of amino acid deriva-
tives in the literature as well as drugs available on the market
(e.g. gabapentin, pregabalin, vigabatrin) (Fig. 2).10–13
part of the design of structures 1–17 was calculation of sev-
eral physicochemical parameters by means of the
Molinspiration online toolkit,15 including molecular weight
(MW), log P, number of hydrogen bond acceptors (nON) and
donors (nOHNH), number of rotatable bonds (nrotb) as well
as topological polar surface area (TPSA) (particular values are
presented in the ESI†). None of the compounds violated more
than one component of the Lipinski “Rule of 5” (MW ≤500,
log P ≤5, H-bond donors ≤5, H-bond acceptors ≤10, nrotb
≤10), which is favorable for potential medicines.16 The calcu-
lated values are also beneficial in terms of blood–brain pene-
tration (according to van de Waterbeemd et al., the favorable
parameters are MW <450, TPSA <90 Å2).17
2. Results and discussion
2.1. Chemistry
All the title compounds were obtained by multistep chemi-
cal synthesis (Scheme 1). Firstly, 2,3-dimethyl-, 2,6-dimethyl-
and 2,4,6-trimethylacetic chlorides (Ia–Ic) were obtained
according to a previously published procedure.8,18 In the next
step, the chlorides were used as acylating agents in the
reaction with appropriate amines in order to obtain the final
compounds 1, 3–7, 12, 13, 15, 16 (reaction carried out in a
biphasic environment) and 2, 14 (reaction carried out in tolu-
ene). For compounds 8–11 and 17 the acetic chlorides were
treated with amino acid methyl ester hydrochlorides in a
biphasic environment to obtain compounds IIa–IId. The
ester groups of the obtained products were subsequently
hydrolysed (compounds 8 and 9) or were subjected to amino-
lysis (compounds 10, 11 and 17).
17 phenoxyacetamide derivatives of phenethylamine, amino-
alkanols or amino acids (Table 1) were designed and
obtained by means of chemical synthesis. Compounds 5–7,
10–12 and 16 were subject to patent protection14 and com-
pounds 1, 2, 4, 9, 13 and 15 possess CAS numbers (however,
their physicochemical properties are unknown). The integral
Fig. 2 Chemical structures of anticonvulsants containing an amino
acid scaffold.
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Table 1 Structures and physicochemical properties of compounds 1–17
Table 1 (continued)
Compd
R
Z
Compd
R
Z
1
2,3-(CH3)2
12
2,6-(CH3)2
2
2,6-(CH3)2
13
14
2,6-(CH3)2
2,6-(CH3)2
3
4
2,6-(CH3)2
2,6-(CH3)2
15
16
2,4,6-(CH3)3
2,4,6-(CH3)3
5
2,6-(CH3)2
6
7
2,6-(CH3)2
2,6-(CH3)2
17
2,4,6-(CH3)3
8
2,6-(CH3)2
The final compounds were purified by crystallization from
organic solvents (hexane, heptane, and toluene). Their identity
and purity were confirmed by means of chromatographic and
spectral analysis (e.g. LCMS, HPLC, IR, 1H NMR, 13C NMR).
For chiral compounds the optical rotation was measured. In
the case of compounds 9 and 13, the plane of rotation of po-
larized light was rotated in the opposite direction compared
to the appropriate amino alcohols used for their synthesis.
9
2,6-(CH3)2
2,6-(CH3)2
10
2.2. Crystallography
The crystal structures of enantiomers 6 and 7 and racemic
compound 12 were studied in order to confirm their struc-
tures and gain information for future design of their poten-
tially active derivatives. An overview of the asymmetric unit of
6, 7 and 12 with the atom numbering is presented in
Fig. 3A, B and C, respectively.
11
2,6-(CH3)2
The crystal structures of 6 and 7 confirm R and S config-
uration at C2, respectively. Both enantiomers crystallize in
the P212121 space group with one molecule in the
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Scheme 1 Synthesis of compounds 1–17.
asymmetric unit. The values of bond distances and angles
are almost the same in both enantiomers. Also in both
enantiomers the disorder of methyl substituents at the ben-
zene ring as well as the carbon atoms C8 and C10 of the
benzene ring is observed.
The crystal structure of 12 confirms its racemic mixture.
This compound crystallizes in the Pna21 space group with one
molecule in the asymmetric unit, and for atoms C1, H1, O1,
and H1O two positions (A for configuration R and B for config-
uration S) with an occupancy factor of 0.5 are observed.
The conformations of molecules are very similar in all the
studied compounds. Two weak intramolecular hydrogen
bonds, N–H⋯O, in which oxygen atoms O1 and O3 are
engaged, seem to have the main influence on the geometry
of the compounds. These hydrogen bonds are observed in all
the presented structures (Table 2). The same type of interac-
tions is present in the similar crystal structures reported
earlier.6,19 The investigated molecules differ only in the sub-
stituents at N1, 1-hydroxyisopropyl in the enantiomers (6 and
7) and 2-hydroxybutyl in 12. Due to a longer hydrocarbon
chain in 12, weak interactions of oxygen atom O1 with C–H
of the aromatic ring of the other molecules in the crystal lat-
tice take place. These interactions appear to provide some
stabilization of the benzene ring with methyl groups in this
crystal structure and no disorder is observed. Similar stabili-
zation effects are not observed in the crystal structures of 6
and 7, and therefore disorder can be observed. The packing
of the molecules in the unit cell can be characterized by the
intermolecular interaction listed in Table 2. The arrangement
of all molecules is dominated by O–H⋯O hydrogen bonds.
The crystal structures are also stabilized by weak C–H⋯O in-
teractions. The nitrogen atom is not involved in any inter-
molecular interactions.
Fig.
3 The molecular structures showing the atom numbering
2.3. Pharmacology
scheme: (A) compound 6; (B) compound 7; (C) compound 12. For
enantiomer S the bonds at C1b are in green. Displacement ellipsoids
are drawn at the 30% probability level.
Pharmacological tests for the title compounds were carried
out within the Epilepsy Therapy Screening Program (ETSP),
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Table 2 Intra- and intermolecular interactions of 6, 7 and 12
H⋯A D⋯A D–H–A
prove an important role of the aminoalkanol substituent for
anticonvulsant activity. They also encourage further research
on amino acid derivatives, with the suggestion that the car-
boxyl group should be significantly modified.
Compd D–H⋯A
(Å)
(Å)
(°)
Symmetry codes
6
N1–H1N⋯O1
2.25
2.20
1.83
2.45
2.691 113
2.596 109
2.697 163
3.372 165
Among active phenoxyacetyl derivatives of aminoalkanols,
aminobutanol (12 – R,S-1-aminobutan-2-ol derivative) and
aminopropanol (4–7) as an amine moiety seem to be
beneficial for activity in MES. Large substituents containing
an aryl ring (compounds 2, 3, 14) seem unprofitable.
Among the derivatives of aminopropanol, both compounds
containing R,S-1-aminopropan-2-ol (4) and R,S-2-amino-
propan-1-ol (5) moieties exhibited comparable activity in the
MES test; however, compound 5 was more toxic, especially af-
ter 4 h. Studies on its enantiomers resulted in compound 6
(derivative of R-2-aminopropan-1-ol), exhibiting the best
pharmacological profile not only among compounds 4–7, but
also among all tested compounds. Our previous studies on
phenoxyalkylamines showed that in the case of aminoalkanols
it is difficult to predict which enantiomer is the more active
one. Thus, it is reasonable to investigate pharmacological
properties of racemic mixture first and then, in case of ob-
served anticonvulsant activity, synthesize the enantiomers.
Compound 1 was more active in MES at a higher dose
(300 mg kg−1) than in the 6 Hz test (100 mg kg−1). The com-
pound differs from compounds 4–7 both with the phenyl ring
substitution (2,3 instead of 2,6) and the presence of a cyclic
aminoalkanol moiety (trans-4-aminocyclohexanol). Both modi-
fications might be responsible for the change in the mecha-
nism of action. Moreover, compounds 6 and 8 were addition-
ally evaluated in the 6 Hz test (mice, i.p.) and compound 6
exhibited moderate (2/4) activity after 15 min at a dose of 120
mg per kg b.w. (see the ESI†). It proves that phenoxyacetyl de-
rivatives of aminoalkanols may be active in both the MES
and the 6 Hz tests and suggests including the 6 Hz test in
future studies as a part of preliminary screening.
The active compounds 4–6, 11 and 12 were subjected to
further pharmacological studies on rats (MES, neurotoxicity,
Table 5). Compound 6 remained the most promising and for
that reason it was chosen for quantitative tests (ED50 = 12.00
mg kg−1, PI >41.640, rats, p.o.) as well as additional screen-
ing for anticonvulsant activity in hippocampal kindling in
rats. Results of the preliminary screening (presented in the
ESI†) encouraged further studies (Table 6). The compound
exhibited activity at a dose of 100 mg kg−1; however, neuro-
toxicity was also observed with this dose.
Previously performed receptor studies for an amine ana-
log of compound 6 (reference compound I, Fig. 1 (ref. 6))
suggested that modulation of the function of the sigma
receptor, serotonergic receptors 5-HT1A and 5-HT trans-
porter, and alpha-adrenergic receptors might be the possible
mechanism of anticonvulsant activity observed within
phenoxyalkyl derivatives of amino alcohols. There is a possi-
bility that this is true also for their amide analogs, as avail-
able pharmacophore models established for binding to the
receptors of interest do not exclude the presence of an
amide moiety.26–28
N1–H1N⋯O3
O1–H1O⋯O2
C9–H9⋯O2
x − 1, y, z
−x + 1, y + 0.5,
−z + 1.5
7
N1–H1N⋯O1
N1–H1N⋯O3
O1–H1O⋯O2
C9–H9⋯O2
2.24
2.16
1.89
2.46
2.688 110
2.591 108
2.688 166
3.369 166
x + 1, y, z
−x + 1, y − 0.5,
−z + 0.5
12
N1–H1N⋯O1A
N1–H1N⋯O1B
N1–H1N⋯O3
2.47
2.42
2.14
2.782 101
2.682
97
2.614 112
2.735 180
2.769 180
3.228 142
O1A–H1OA⋯O2 1.89
O1B–H1OB⋯O2 1.92
x, y, z − 1
x, y, z − 1
C9–H9⋯O1B
2.44
−x + 0.5, y − 0.5,
z + 0.5
C11–H11⋯O1A 2.70
3.563 155
−x + 0.5, y + 0.5,
z + 0.5
Epilepsy Branch, National Institute of Neurological and Com-
municative Disorders and Stroke, NIH, Rockville, MD, USA.20
All achieved compounds were the subject of preliminary eval-
uation of their anticonvulsant activity and neurological toxic-
ity (rotarod) in mice after i.p. administration, including MES
and scMET (compounds 2–13 and 15–17, Table 3) or 6 Hz (44
mA (ref. 21)) and MES (compounds 1, 14, Table 4) as models
of convulsions. Among all tested compounds, 1, 4–6, 11 and
12 showed activity in preliminary screening in either the MES
or the 6 Hz test and some of them were subjected to further
pharmacological studies (Tables 5–7). None of the tested
compounds was active in scMET, which evaluates the ability
of protecting from clonic, forebrain seizure22 and the positive
results may correlate with potential utility against absence
seizures.23
Previously, we obtained and screened for anticonvulsant
activity amine analogs of compounds 3–7, 12, 13 and 16.4–7
Their amide derivatives presented in this paper showed
weaker activity in all cases. However, some of them still pos-
sessed notable pharmacological profiles (4, 5, 6, 12) and they
were also generally less neurotoxic in rotarod compared to
their amine analogs. For instance, the most promising com-
pound among the achieved amides, compound 6, possessed
a lower value of protective index in the MES test performed
on mice after i.p. administration than its amine analog (1,43
and 5,52, respectively) but higher in the rat model after p.o.
administration (>41.64 and >17.48, respectively). The out-
comes indicate that although the amine moiety seems to be
preferential in terms of anticonvulsant activity, in the case of
obtaining a potent amine showing high neurotoxicity in ani-
mal tests, it might be reasonable to explore the pharmacolog-
ical activity of its amide derivatives.
Most of the active compounds possessed aminoalkanol as
an amide moiety. The exception was compound 11 con-
taining an N-methylamide derivative of alanine. These results
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Table 3 Anticonvulsant properties of compounds 1–17 in MES (50 mA) and scMET as well as their neurotoxicity in the rotarod test (mice, i.p.)
MESa
scMETb
TOXb
Time [h]
0.25
Dose
Compd
[mg kg−1
]
0.5
1.0
4.0
0.25
0.5
1.0
4.0
0.25
0.5
1.0
4.0
2
30
/
/
/
/
/
/
/
0/1
0/3
0/1
0/1
0/3
0/1
0/1
0/3
1/1
0/1
0/3
1/1
0/1
0/3
0/4
/
/
/
/
/
/
/
/
0/1
0/3
0/1
0/1
0/3
0/1
0/1
0/3
0/1
0/1
0/3
0/1
0/1
0/3
0/4
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
/
/
/
/
/
/
/
/
0/4
0/8
1/4
0/4
1/8
0/4
0/4
0/8
4/4
0/4
2/8
4/4
0/4
0/8
/
/
/
/
/
/
/
/
0/2
0/4
0/2
0/2
0/4
0/2
0/2
0/4
0/2
0/2
2/4
0/2
0/2
0/4
/
100
300
30
100
300
30
100
300
30
100
300
30
3
4
5
6
2/3
/
/
2/3
/
/
2/3
4/4
/
0/3
/
/
0/3
/
/
0/3
0/4
/
1/3
/
/
1/3
/
/
0/3
/
8/8
/
0/3
/
/
0/3
/
/
0/3
/
0/8
/
100
110
220
300
/
/
1/8
2/4
0/8
0/2
/
1/1
/
0/1
0/1
0/1
TPE [h] = 0.25
MES ED50 = 97.93 (78.524–112.599) mg kg−1
TD50 = 139.96 (128.762–156.811) mg kg−1
PI = 1.429
7
30
/
0/1
0/3
1/1
0/1
0/3
0/1
0/1
0/3
0/1
0/1
0/3
1/1
0/1
2/3
1/1
0/1
0/3
1/1
0/1
0/3
1/1
0/1
0/3
1/1
0/1
0/3
0/1
0/1
0/3
1/1
/
0/1
0/3
1/1
0/1
0/3
0/1
0/1
0/3
0/1
0/1
0/3
0/1
0/1
0/3
0/1
0/1
0/3
0/1
0/1
0/3
0/1
0/1
0/3
0/1
0/1
0/3
0/1
0/1
0/3
0/1
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
—
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
1/5
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
/
1/4
3/8
1/4
0/4
0/8
0/4
0/4
0/8
0/4
0/4
2/8
1/4
0/4
1/8
4/4
0/4
0/8
4/4
0/4
0/8
4/4
0/4
1/8
2/4
0/4
0/8
4/4
0/4
0/8
1/4
/
0/4
1/4
1/2
0/2
0/4
0/2
0/2
0/4
0/2
0/2
0/4
0/2
0/2
0/4
0/2
0/2
0/4
0/2
0/2
0/4
0/2
0/2
0/4
0/2
0/2
0/4
0/2
0/2
0/4
0/2
100
300
30
100
300
30
100
300
30
100
300
30
100
300
30
100
300
30
100
300
30
100
300
30
1/3
/
/
/
/
/
/
/
1/3
/
/
/
/
/
/
/
1/3
/
/
/
/
/
/
/
1/3
/
/
/
/
/
/
/
8
9
10
11
12
13
15
16
17
/
/
/
/
0/3
/
/
/
/
/
3/3
/
/
1/3
/
/
0/3
/
/
/
/
/
0/3
/
/
/
/
/
0/3
/
/
0/3
/
/
0/3
/
/
/
/
/
0/3
/
/
/
/
/
1/3
/
/
1/3
/
/
0/3
/
/
/
/
/
1/3
/
/
/
/
/
0/3
/
/
0/3
/
/
0/3
/
/
/
/
/
100
300
30
100
300
—
0/3
/
0/3
/
0/3
/
0/3
/
CBZ24
ED50 = 7.81 (6.32–8.45) mg kg−1
TD50 = 45.4 (32.9–54.4) mg kg−1
Allopregnanolone25
—
—
TPE = 0.17 [h]
—
ED50 = 13.7 (10.1–18.7) mg kg−1
a
b
Number of animals protected/number of animals tested. Number of animals exhibiting toxicity/number of animals tested in the rotarod
test. / not tested; — no data.
2.4. In vitro cytotoxicity studies
ing in vitro cytotoxicity tests on astrocytes. The astrocyte
model was chosen due to the previously observed influence
of known anticonvulsants (used in higher concentrations) on
cells originating from the nervous system.29 Compound 6
Compound 6, possessing the most favorable pharmacological
profile, was also subjected to further toxicity studies, includ-
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Table 4 Anticonvulsant properties of compounds 1 and 14 in 6 Hz (44
mA) and MES (50 mA) as well as their neurotoxicity in the rotarod test
(mice, i.p.)
Table 6 Activity in hippocampal kindling for compound 6 and its neuro-
toxicity in rats, i.p.
Dose
Seizure
Duration Test results
Compd [mg kg−1
]
Time [h] score SEMa [s] SEM (after 0.25 h)
6a Hz
MESa
TOXb
Time [h]
6
50
0
15
45
4.4 0.2
4.9 0.1
4.9 0.1
5.0 0.0b
4.9 0.1
4.9 0.1
5.0 0.0
3.6 7b
4.7 0.2
4.9 0.1
4.7 0.2
4.9 0.1
72
71
73
75
77
72
68
5
5
4
6
7
7
5
0/7c,d
Dose
Compd
[mg kg−1
]
0.5
2.0
0.5
2.0
0.5
2.0
1
30
0/4
2/4
3/4
0/4
0/4
0/4
0/4
0/4
4/4
0/4
0/4
1/4
0/4
0/4
4/4
0/4
1/4
0/4
ED50 = 7.81
(6.32–8.45)
mg kg−1
0/4
0/4
1/4
0/4
0/4
1/4
0/8
0/8
1/8
0/8
0/8
0/8
TD50 = 45.4
(32.9–54.4)
mg kg−1
0/8
0/8
0/8
0/8
0/8
0/8
75
100
300
30
100
300
—
105
135
0
15
45
75
105
135
2/7c
3/7d
14
100
48 10
86
80
77
76
8
6
6
6
CBZ24
Max. 75%
protection
at 40 and
80 mg kg−1
a
b
c
Racine's scale. Significantly different from control. No. animals
a
b
d
Number of animals protected/number of animals tested. Number
of animals exhibiting toxicity/number of animals tested in the
rotarod test. — no data.
protected/tested. Number of animals exhibiting toxicity/number of
animals tested.
showed no cytotoxicity in concentrations ranging from 10 to
200 μM in the MTT assay (both mice and human astrocytes)
and neutral red test (mice astrocytes). Moreover, the
compound did not significantly affect mice astrocyte prolifer-
ation (CV assay). Taking the above into account, it can be
claimed that it is completely safe for cells that are derived
from the nervous system. For a graphical interpretation of
the presented results, see the ESI.†
compounds demonstrated values of Clint lower than those
reported for imipramine30 or propranolol.31 It is worth
noting that compound 12, which was the more lipophilic one
(clog P = 2.33), possessed a higher Clint value than that of the
more hydrophilic compound 6.32
In both compounds (6 and 12) the predominant transfor-
mation is hydroxylation of the parent compound (Table 8).
Both metabolites have a protonated molecular ion [M + H]+
with a molecular weight 16 Da higher than those of the
parent compounds.
2.5. Metabolic stability
According to the results presented in Table 7, compound 6
can be classified as metabolically stable, as its rat intrinsic
clearance is lower than 10 μl mg−1 min−1, while compound
12 exhibited moderate in vitro metabolic stability. Both
2.6. Mutagenicity
2.6.1. Assessment of mutagenic properties of test com-
pounds. As a part of our broader studies on mutagenicity in
Table 5 Anticonvulsant activity in MES (150 mA) of compounds 4–6, 11 and 12 and their neurotoxicity evaluation performed on rats
Rats, p.o. (dose = 30 mg kg−1
)
Rats, i.p.
Time [h]
Time [h]
0.25
Dose
0.25
0.5
1.0
2.0
4.0
0.5
1.0
2.0
4.0
Compd
Test
Test
[mg kg−1
]
4
MES
TOX
MES
TOX
MES
TOX
0/4
0/4
2/4
0/4
2/4
0/4
0/4
0/4
2/4
0/4
4/4
0/4
0/4
0/4
0/4
0/4
1/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
1/4
0/4
2/4
0/4
1/4
0/4
—
5
6
—
TOX
30
100
300
1/2
1/2
2/2
0/2
0/2
2/2
0/2
0/2
2/2
0/2
0/2
2/2
0/2
0/2
2/2
Quantitative assay
MES ED50 = 12.00 (7.098–20.671)
TD50 > 500.00
PI > 41.640
TPE [h] = 0.50
11
MES
TOX
MES
TOX
MES
TOX
1/4
0/4
3/4
0/4
1/4
0/4
4/4
0/4
0/4
0/4
1/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
—
12
TOX
—
100
4/4
4/4
0/4
/
/
CBZ24
ED50 = 5.35 (3.26–7.62) mg kg−1
TD50 = 364 (223–500) mg kg−1
Results presented as: number of animals protected/number of animals tested (MES) or number of animals exhibiting toxicity/number of
animals tested (TOX). / not tested; — no data.
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Table 7 Stability (t1/2 and Clint) of 6 and 12 in the rat liver microsomal
system
2.6.2. Antimutagenicity evaluation of test compounds. In
the antimutagenicity assay performed with S. typhimurium
TA100 and TA1535 strains, compound 3 exhibited the highest
antimutagenic effect against sodium azide mutagenicity at
various test concentrations (Table 9). This compound demon-
strated strong inhibition rates of NaN3 mutagenicity (41–
58%) for three test concentrations in strain TA100 and two
concentrations in strain TA1535. Compound 6 exhibited high
or medium antimutagenic activities (inhibition rates from
29% to 57%) on NaN3-induced mutagenesis in all tested con-
centrations in both Salmonella strains. Compound 13 re-
duced strongly or moderately sodium azide mutagenicity only
in the TA100 strain; in TA1535, low antimutagenic effect was
observed.
Protein
concentration
Clint
Compd
(mg ml−1
)
clog P t1/2 (min)
(μl mg−1 min−1
)
6
12
0.4
0.4
1.80
2.33
2.75
5.04
182.4
30.4
—
9.5
57
79 (ref. 31)
Propranolol 0.5 (ref. 31)
Imipramine 0.5 (ref. 30)
4.6 (ref. 30) 302.0 (ref. 30)
Table 8 Summary of 6 and 12 metabolites generated in rat liver micro-
somes after 60 min
Compd Metabolite Mass shift (Da) m/z Retention time (min)
6
Parent
M1
Parent
M1
0
+16
0
238 4.73
254 3.38
252 5.15
268 3.73
3. Conclusions
12
+16
Within the presented research we designed and synthesized
17 substituted phenoxyacetamides, including mainly amide
analogs of the previously achieved centrally active amines4–7
and several derivatives of (un)modified amino acids. The
anticonvulsant activity of the obtained compounds was evalu-
ated within the Epilepsy Therapy Screening Program (ETSP)20
with use of the following animal models: MES, 6 Hz, scMET,
hippocampal kindling, neurotoxicity (rotarod) performed on
mice and/or rats. The results indicated compound 6 as the
one possessing the most favorable pharmacological profile
and allowed us to draw several conclusions.
a group of phenoxyalkyl and phenoxyacetyl derivatives of
amines,6,33 compounds 3, 6, 12 and 13 were examined in
the Ames test. They did not produce any mutagenic effect in
the employed S. typhimurium TA100 and TA1535 strains. At
the concentrations of 500, 250, 100 and 50 μg per plate of
test compounds, the mutagenicity index (MI) ranged from
0.7 to 1.7, suggesting a lack of mutagenic activity of the
studied compounds both with and without metabolic activa-
tion. The mean number of revertants
and mutagenicity indices for all tested concentrations are
available in the ESI.†
standard deviation
In general, amide derivatives di- or trisubstituted with
methyl groups in the phenyl ring seem to be less promising
compared to their amine analogs. However, due to their
Table 9 Antimutagenicity results of compounds 3, 6, 12 and 13 in Salmonella typhimurium assay
Number of revertants
S. typhimurium
TA 100
TA 1535
Concentration
Compd
(μg per plate)
Mean S.D.
Inhibition (%)
Mean S.D.
Inhibition (%)
Negative control
Positive control
3
—
5
102
8
—
—
10
4
—
—
533 36
355 11
313 17
340 14
460 12
360 29
371 27
346 20
288 15
332 13
484 25
422 34
443 17
348 17
413 19
385 22
420 25
397 25
345 26
261 21
222 11
500
250
100
50
500
250
100
50
500
250
100
50
500
250
100
50
41c
51c
45c
17a
40b
38b
43c
57c
47c
11a
26b
21a
43c
28b
34b
26b
13a
35b
45c
58c
31b
47c
43c
29b
27b
56c
51c
42c
24a
19a
22a
12a
171
5
6
276 18
217 21
231 14
285
292 13
182
198 10
234 12
305
9
12
13
4
2
323 24
313
350
4
4
*Mean S.D. – mean value standard deviation and mutagenicity inhibition are given; negative control – dimethyl sulfoxide (DMSO); positive
b
c
control – sodium azide (NaN3, 5 μg per plate).a No antimutagenic effect (<25% inhibition). Moderate effect (25–40% inhibition). Strong
antimutagenic effect (>40% inhibition).
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relative safety, in some cases it might be reasonable to inves-
tigate the amide analogs of potent amines. Incorporation of
the amino acid moiety did not result in increase of activity.
Modification of a carboxyl group might result in better phar-
macological properties of the designed compounds; however,
further studies on this subject should be conducted in order
to draw proper conclusions. For future studies, inclusion of
the 6 Hz test instead of the scMET test as preliminary screen-
ing of anticonvulsant activity seems to be reasonable in the
case of the studied group of compounds.
University (Cracow, Poland) using a Bruker Avance II spectro-
meter at 500.13 MHz (1H NMR for 2–4, 12), 300 MHz
(1H NMR for 11) and 125.17 MHz (13C NMR for 2 and 3), or
at Jagiellonian Centre of Innovation (NMR Laboratory, Cra-
cow, Poland) using a Bruker Avance III HD spectrometer op-
erating at 400.17 MHz and 100 MHz (1H and 13C NMR for 7,
respectively). The following abbreviations are used in spectra
description: singlet (s), doublet (d), triplet (t), multiplet (m),
and broad signal (br. s.).
Measurement of optical rotation ([α]25809.0) for chiral com-
pounds was carried out using a Jasco 2000 system (2% meth-
anol solutions).
4. Experimental protocols
4.1.1. General procedure for preparation of compounds
1–17. Compounds 1–17 were obtained via multistep chemical
synthesis. In the first step, the appropriately substituted
phenoxyacetic chlorides (Ia–Ic) were obtained according to a
previously published procedure.8,18 2,3-Dimethyl-, 2,6-
dimethyl- or 2,4,6-trimethylphenol (0.2 mol) was dissolved in
100 ml of 2 M NaOH aqueous solution and heated in a round
bottom flask under a reflux condenser while an equimolar
amount of 2-chloroacetic acid dissolved in 100 ml of 2 M
NaHCO3 aqueous solution was added dropwise. After 1 hour
of heating, activated carbon was added and the product was
obtained by filtering the reaction mixture using fluted filter
paper and subsequently acidifying the obtained filtrate.
Then, the phenoxyacetic acids were transformed into chlo-
rides by heating in a round bottom flask under a reflux con-
denser for about 1 h with an excess of thionyl chloride (molar
ratio 1 : 1.5) and in benzene solution. The unreacted thionyl
chloride and solvent were then distilled off under vacuum
and the acid chloride remaining in the flask was diluted with
toluene. The obtained products Ia and Ib are known com-
pounds of reagent grade, while compound Ic was previously
described.34
In the next step, the appropriate aminoalkanol (1, 3–7, 12,
13, 16), amine (2) or amino acid methyl ester (IIa–IId) (0.02
mol) was dissolved in 50 ml of 1 M K2CO3 aqueous solution
and subsequently about 30 ml of toluene was added. The
mixture was placed on a magnetic stirrer at 10–12 °C and an
equimolar amount of the appropriate acid chloride in toluene
was added dropwise for 2 h. All reagents were stirred for an
additional 10 min after adding the whole amount of acid
chloride, and then heated to the boiling point and left to
cool. The precipitated amide deposit was filtered off, stirred
4.1. Chemistry
The reagents and solvents for synthetic purposes were com-
mercially available materials of reagent grade and were pur-
chased from Alfa Aesar (purchased from Chemat, Gdansk,
Poland), Sigma-Aldrich (purchased from Sigma-Aldrich,
Poznan, Poland) or Merck Sp. z o.o. (Warszawa, Poland). The
solvents used for LCMS and HPLC were of LCMS grade and
were purchased from Avantor Performance Materials Poland
S.A., Gliwice, Poland.
The physicochemical parameters for the designed struc-
tures were calculated in silico using the ChemBioDraw Ultra
14.0 program. Melting points (mp) are uncorrected and were
determined using a Büchi SMP-20 apparatus.
Analyses of C, H and N were within 0.4% of the theoreti-
cal values. The IR spectra were recorded on a Jasco FT/IR 410
spectrometer (KBr pellets).
The LCMS system consisted of a Waters Acquity UPLC sys-
tem coupled to a Waters TQD mass spectrometer (electro-
spray ionization mode ESI-tandem quadrupole). All the analy-
ses were carried out using an Acquity UPLC BEH C18, 1.7 lm,
2.1 × 100 mm column. A flow rate of 0.3 ml min−1 and a gra-
dient of 5–95% B over 10 min and then 100% B over 2 min
was used. Eluent A: water/0.1% HCO2H; eluent B: acetoni-
trile/0.1% HCO2H. LCMS data were obtained by scanning the
first quadrupole in 0.5 s in a mass range from 50 to 1000 Da;
eight scans were summed up to produce the final spectrum.
Analytical HPLC analysis was carried out with an LC Ulti-
mate 3000 system consisting of a pump, a degasser, an
autosampler and a column heater, equipped with a Corona
charged aerosol detector (Corona CAD). Nitrogen from an
NG-4-1 nitrogen generator set at 35 psi was introduced to the
detector. Data were processed with Chromeleon 6.8 software.
The following columns were used: (A) Allure PFP (150 mm ×
4.6 mm; 5 μm particle size); (B) Hypersil Gold C18 (150 mm
× 4.6 mm; 5 μm particle size). The isocratic elution was
performed with 30% water (solvent A) and 70% methanol
(solvent B) at the temperature of 20 °C. The flow rate was set
at 1.0 ml min−1, and the injection volume was 20 μl.
with
a 10% solution of NaHCO3 and (after drying)
recrystallized from a heptane–toluene (1 : 1) mixture. Com-
pounds IIa–IId are semi-products for synthesis compounds
8–11 and 17. The physicochemical parameters of IId are
presented below, while compounds IIa–IIc were used as raw
products after washing with a 10% solution of NaHCO3.
Compound IIc is commercially available (CAS 1994257-51-0).
For compound 15, the reaction with the appropriate
aminoalkanol was carried out in toluene, in the presence
of K2CO3 as a proton acceptor. The reagents (0.03 mol of
trans-4-aminocyclohexan-1-ol, 0.06 mol of 2,6-dimethyl-
phenoxyacetic acid chloride, and 0.03 mol of K2CO3) were
NMR spectra were recorded at the Faculty of Pharmacy,
Jagiellonian University Medical College (Cracow, Poland) with
a Varian Mercury-VX 300 NMR spectrometer operating at 300
MHz (1H NMR for 1, 5, 6, 8–10, 13–17) or 75 MHz (13C NMR
for 1, 4–6, 8–17) at the Faculty of Chemistry, Jagiellonian
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heated in a round bottom flask under a reflux condenser.
After about 6 h the inorganic salt was filtered off, the
remaining mixture was concentrated and the obtained prod-
uct was crystallized from hexane.
In order to obtain compounds 8–11 and 17, the ester
groups of compounds IIa–IId were hydrolysed with 10%
NaHCO3 (8 and 9) at room temperature (6–8 h) or were
Ar–(CH3)2); δ 13C NMR (DMSO-d6): 167.79 (>CO), 154.52
(Ar–C1), 143.45 (Ar′–C1), 130.33 (Ar–C2,6), 128.82 (Ar′–C3,5),
128.00 (Ar–C3,5), 127.09 (Ar′-2,6), 126.07 (Ar′–C4), 124.25 (Ar–
C4), 70.98 (>CH–Ar′), 70.39 (–CH2–CO–), 46.21 (–NH–CH2–),
15.87 (Ar–(CH3)2); LCMS, m/z: 300.19 (M+ + 1, 98.26%); HPLC,
RT (A) = 8.56 min, 97.62%; found: C 72.20, H 7.07, N 4.70,
calc. for C18H21NO3: C 72.10, H 7.21, N 4.46.
subjected to aminolysis with use of NH3(aq
(10, 17) or
4 . 1 . 1 . 4 .
R , S - 2 - ( 2 , 6 - D i m e t h y l p h e n o x y ) - N - ( 2 -
conc)
methylamine (11) with the addition of methanol. The precipi-
tated final products were recrystallized from a hexane–tolu-
ene (1 : 1) mixture.
hydroxypropyl)acetamide (4). (62%); mp 106–108; C13H19NO3;
IR (KBr) ν = 3421, 3340, 2962, 2922, 2875, 1655, 1541, 1473,
1194, 1140, 1051, 972, 791, 665, 579, 436 cm−1; δ 1H NMR
(DMSO-d6): 7.99 (1H, t, J = 5.6 Hz, NH), 7.03 (2H, d, J = 7.5
Hz, Ar–H), 6.95 (1H, t, J = 7.5 Hz, Ar–H), 4.79 (1H, d, J = 5.1
Hz, OH), 4.21 (2H, s, Ar–O–CH2–), 3.71–3.79 (1H, m, >CH–
OH), 3.15–3.22 (1H, m, –CHH–NH–), 3.08–3.14 (1H, m, –CH
H–NH–), 2.26 (6H, s, Ar–(CH3)2), 1.06 (3H, d, J = 6.2 Hz,
>CH–CH3). δ 13C NMR (DMSO-d6): 168.3 (>CO), 155.1
(Ar–C1), 130.8 (Ar–C2,6), 129.3 (Ar–C3,5), 124.7 (Ar–C4), 71.0
(–CH2–CO–), 65.4 (>CH–), 46.4 (–CH2–CH<), 21.5 (>CH–
CH3), 16.4 (Ar–(CH3)2); LCMS, m/z: 238.12 (M+ + 1, 100%);
HPLC, RT (A) = 4.93 min, 97.19%. CAS 11567-97-47-5.
4.1.1.5. R,S-2-(2,6-Dimethylphenoxy)-N-(1-hydroxypropan-2-
yl)acetamide (5). (60%); mp 98–100; C13H19NO3; δ 1H NMR
(CDCl3): 6.92–7.13 (4H, m, Ar–H3,4,5, NH), 4.35 (1H, s, OH),
4.30 (1H, s, Ar–O–CHH–), 4.29 (1H, s, Ar–O–CHH–), 4.14–4.25
(1H, m, –CH<), 3.59–3.81 (2H, m, –CH2–OH), 2.67–2.75 (1H,
m, OH), 2.26 (6H, s, Ar–(CH3)2), 1.29 (3H, d, J = 7.0 Hz); δ
13C NMR (CDCl3): 169.3 (>CO), 154.4 (Ar–C1), 130.4 (Ar–
C2,6), 129.1 (Ar–C3,5), 124.7 (Ar–C4), 70.4 (–CH2–CO–), 66.6
(–CH2–OH), 47.4 (>CH–), 17.1 (>CH–CH3), 16.3 (Ar–(CH3)2);
LCMS, m/z: 238.18 (M+ + 1, 99.08%); HPLC, RT (A) = 5.40
min, 100%.
The purity and identity of the obtained compounds
1
were confirmed by means of LCMS, HPLC, IR, H NMR and
13C NMR.
Characterisation of semi-product
R,S-Methyl 2-(2-(mesityloxy)acetamido)butanoate (IId). (80%);
mp 64–66 °C; C15H21NO4; LCMS, m/z: 294.14 (M+ + 1),
100.00%.
Characterisation of final products
4 . 1. 1. 1.
trans-2 -(2, 3 -Dimethylphenoxy)-N-(4-
hydroxycyclohexyl)acetamide (1). (60%); mp 177–179 °C;
C16H23NO3; δ 1H NMR (CDCl3): 6.99–7.11 (1H, m, Ar–H5),
6.85 (1H, d, J = 7.57 Hz, Ar–H4), 6.64 (1H, d, J = 8.21 Hz, Ar–
H6), 6.41 (1H, d, J = 8.21 Hz, NH), 4.44 (2H, s, CH2), 3.77–
3.97 (1H, m, CH–OH), 3.55–3.72 (1H, m, CH–N), 2.29 (3H, s,
Ar–CH3), 2.18 (3H, s, Ar–CH3), 1.93–2.12 (4H, m, cyclohex–
H3,5), 1.56 (1H, br s, OH), 1.35–1.52 (2H, m, cyclohex–H2,6),
1.18–1.35 (2H, m, cyclohex–H2,6); δ 13C NMR (CDCl3): 168.0
(>CO), 155.3, (Ar–C1), 138.4 (Ar–C3), 126.2 (Ar–C5),
125.0 (Ar–C4), 123.8 (Ar–C2), 109.7 (Ar–C6), 69.5 (>CH–OH),
68.1 (–CH2–CO–), 47.1 (–NH–CH<), 33.7 ((CH2)2 > CH–OH),
30.7 ((CH2)2 > CH–NH–), 20.1 (Ar–CH3IJ3)), 11.9 (Ar–CH3IJ2));
LCMS, m/z 278.112 (M+ + 1, 97.70%); HPLC, RT (B) = 3.63
min, 99.15%. CAS 1157017-49-6.
4.1.1.6. R-(−)-2-(2,6-Dimethylphenoxy)-N-(1-hydroxypropan-2-
yl)acetamide (6). (62%); mp 122–124; C13H19NO3; δ 1H NMR
(DMSO-d6): 7.75 (1H, d, J = 8.2 Hz, NH), 6.98–7.04 (2H, m, Ar–
C3,5), 6.88–6.95 (1H, m, Ar–C4), 4.75 (1H, t, J = 5.6 Hz, OH),
4.17 (1H, s, Ar–O–CHH–), 4.16 (1H, s, Ar–O–CHH–), 3.90 (1H,
dd, J = 7.6; 6.4 Hz, >CH–), 3.32–3.45 (2H, m, –CH2–OH), 2.20
(6H, s, Ar–(CH3)2), 1.08 (3H, d, J = 7.0 Hz, >CH–CH3); δ 13C
NMR (DMSO-d6): 167.7 (>CO), 155.4 (Ar–C1), 130.8 (Ar–
C3,5), 129.3 (Ar–C2,6), 124.6 (Ar–C4), 71.2 (–CH2–CO–), 64.6
(–CH2–OH), 46.7 (>CH–), 17.5 (>CH–CH3), 16.4 (Ar–(CH3)2);
4.1.1.2. 2-(2,6-Dimethylphenoxy)-N-phenethylacetamide (2).
1
(54%); mp 84–86 °C; C18H21NO2; δ H NMR (DMSO-d6): 8.20
(1H, t, J = 5.6 Hz, NH), 7.27–7.33 (2H, m, Ar′–H2,6), 7.18–7.27
(3H, m, Ar′–H3,4,5), 7.04 (2H, dddd, J = 8.2 Hz; 6.8 Hz; 1.4
Hz; 0.8 Hz, Ar–H3,5), 6.95 (1H, dd, J = 8.2 Hz; 6.8 Hz, Ar–H4),
4.16 (2H, s, Ar–O–CH2), 3.43 (2H, dt, J = 5.6 Hz; 7.5 Hz, –NH–
CH2), 2.81 (2H, t, J = 7.5 Hz, –CH2–Ar), 2.19 (6H, s,
Ar–(CH3)2); δ 13C NMR (DMSO-d6): 167.66 (>CO), 154.57
(Ar–C1), 139.33 (Ar′–C1), 130.35 (Ar–C2,6), 128.80 (Ar′–C3,5),
128.68 (Ar–C3,5), 128.29 (Ar′-2,6), 126.09 (Ar′–C4), 124.22 (Ar–
C4), 70.47 (–CH2–CO–), 39.76 (–NH–CH2–), 35.01 (–CH2–Ar′),
15.83 (Ar–(CH3)2); LCMS, m/z 284.17 (M+ + 1, 97.57%); HPLC,
RT (B) = 6.67 min, 97.08%. CAS 447437-41-4.
LCMS, m/z: 238.22 (M+ + 1, 100%); HPLC, RT (A) = 5.40 min,
20.0
589
99.78%; [α]
= −1.4104° (2% concentration in MeOH).
4.1.1.7. S-(+)-2-(2,6-dimethylphenoxy)-N-(1-hydroxypropan-2-
yl)acetamide (7). (60%), mp 121–123; C13H19NO3; δ 1H NMR
(CDCl3): 7.08–7.16 (1H, m, NH), 6.96–7.08 (3H, m, Ar–H3,4,5),
4.26–4.37 (2H, m, Ar–O–CH2–), 4.22 (1H, m, –CH<), 3.77 (1H,
dd, J = 10.76; 3.13 Hz, –CHH–OH), 3.66 (1H, dd, J = 11.00; 5.92
Hz, –CHH–OH), 3.00 (1H, br s, OH), 2.28 (6H, s, Ar–(CH3)2),
1.31 (3H, d, J = 6.85 Hz, >CH–CH3); δ 13C NMR (CDCl3):
169.39 (>CO), 154.41 (Ar–C1), 130.38 (Ar–C3,5), 129.17 (Ar–
C2,6), 124.77 (Ar–C4), 70.41 (–CH2–CO–), 66.95 (–CH2–OH),
47.58 (>CH–), 17.06 (>CH–CH3), 16.28 (Ar–(CH3)2); LCMS, m/
4.1.1.3. R,S-2-(2,6-Dimethylphenoxy)-N-(2-hydroxy-2-
phenylethyl)acetamide (3). (49%); mp 113–114; C18H21NO3; IR
(KBr) ν = 3396, 3317, 2939, 2871, 1635, 1556, 1479, 1196,
1
1030, 762 cm−1; δ H NMR (DMSO-d6): 7.96 (1H, t, J = 5.6 Hz,
NH), 7.31–7.40 (4H, m, Ar′–H2,3,5,6), 7.18–7.27 (1H, m, Ar′–
H4), 7.03 (1H, d, J = 7.0, Ar–H), 6.95 (1H, d, J = 7.0 Hz, Ar–H),
6.93 (1H, d, J = 7.0 Hz, Ar–H), 5.57 (1H, d, J = 4.5 Hz, OH),
4.72–4.78 (1H, m, CH–OH), 4.17 (2H, s, Ar–O–CH2), 3.41–3.48
(1H, m, N–CHH), 3.29–3.34 (1H, m, N–CHH), 2.19 (6H, s,
z: 238.18 (M+ + 1, 100%); HPLC, RT (A) = 5.38 min, 99.50%;
20.0
589
[α]
= 1.2198° (2% concentration in MeOH).
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4.1.1.8.
R,S-(2-(2,6-Dimethylphenoxy)acetyl)alanine
(8).
CH3); LCMS, m/z: 252.21 (M+ + 1, 100.00%); HPLC, RT (A) =
5.94 min, 100%.
1
(70%), mp 180–182; C13H17NO4; δ H NMR (CDCl3): 7.49 (1H,
d, J = 7.6 Hz, NH), 6.94–7.05 (3H, m, Ar–H3,4,5), 4.76 (1H, m,
>CH–), 4.26–4.41 (2H, m, Ar–O–CH2–), 2.27 (6H, s,
Ar–(CH3)2), 1.58 (3H, d, J = 7.0 Hz, >CH–CH3); δ 13C NMR
(CDCl3): 176.4 (–COOH), 169.2 (>CO), 154.3 (Ar–C1), 130.4
(Ar–C2,6), 129.2 (Ar–C3,5), 124.8 (Ar–C4), 70.1 (–CH2–CO–),
47.7 (>CH–), 18.2 (>CH–CH3), 16.3 (Ar–(CH3)2); LCMS, m/z:
252.14 (M+ + 1, 98.99%); HPLC, RT (C) = 2.252 min.
4.1.1.13. S-(−)-2-(2,6-Dimethylphenoxy)-N-(1-hydroxybutan-2-
yl)acetamide (13). (70%), mp 76–78; C14H21NO3; δ 1H NMR
(CDCl3): 6.90–7.10 (3H, m, Ar–H3,4,5), 4.23–4.39 (2H, m, Ar–
O–CH2–), 3.99 (m, 1H, –CH<), 3.65–3.83 (2H, m, –CH2–OH),
2.56–2.64 (1H, m, impurity), 2.27 (6H, s, Ar–(CH3)2), 1.58–1.76
(2H, m, –CH2–CH3), 1.03 (3H, t, J = 7.6 Hz, –CH2–CH3); δ 13C
NMR (CDCl3): 169.5 (>CO), 154.3 (Ar–C1), 130.4 (Ar–C2,6),
129.1 (Ar–C3,5), 124.7 (Ar–C4), 70.4 (–CH2–CO–), 64.8 (–CH–
OH), 53.0 (>CH–), 24.2 (–CH2–CH3), 16.2 (Ar–(CH3)2), 10.5
4.1.1.9. S-(−)-(2-(2,6-Dimethylphenoxy)acetyl)alanine (9).
(68%); mp 188–190; C13H17NO4; δ H NMR (CDCl3): 7.47 (1H,
1
d, J = 7.0 Hz, NH), 6.93–7.06 (3H, m, Ar–H3,4,5), 4.76 (1H, m,
>CH–), 4.26–4.41 (2H, m, Ar–O–CH2–), 2.27 (6H, s,
Ar–(CH3)2), 1.58 (3H, d, J = 7.0 Hz, >CH–CH3); δ 13C NMR
(CDCl3): 176.2 (–COOH), 169.2 (>CO), 154.3 (Ar–C1), 130.4
(Ar–C2,6), 129.2 (Ar–C3,5), 124.8 (Ar–C4), 70.2 (–CH2–CO–),
(–CH2–CH3); LCMS, m/z: 252.14 (M+ + 1, 97.35%); HPLC,
20.0
RT (A)
=
5.58 min, 99.05%; [α]
=
−14.1980° (2%
589
concentration in MeOH). CAS 1983486-85-6.
4.1.1.14. trans-4-(2-(2,6-Dimethylphenoxy)acetamido)cyclohexyl
2-(2,6-dimethylphenoxy)acetate (14). (52%), mp 170–171;
1
47.7 (>CH–), 18.2 (>CH–CH3), 16.3 (Ar–(CH3)2); LCMS, m/z:
C26H33NO5; δ H NMR (CDCl3): 6.92–7.06 (6H, m, Ar–H), 6.82
252.14 (M+ + 1, 98.61%); [α]
= −9.8930° (2% concentration
(1H, d, J = 8.2 Hz, NH), 4.93 (1H, tt, J = 10.6, 4.0 Hz, CH–O),
4.41 (2H, s, CO–CH2–O–Ar), 4.28 (2H, s, CH2–CO–NH), 3.87–
4.06 (1H, m, NH–CH), 2.31 (6H, s, Ar–CH3 (amide)), 2.26 (6H,
s, Ar–CH3 (ester)), 2.14 (4H, ddd, J = 13.3 Hz, 9.6 Hz, 3.8 Hz,
4H, cyclohex), 1.60–1.71 (2H, m, J = 3.1 Hz, cyclohex), 1.59
(overlap – H2O from CDCl3), 1.36–1.52 (2H, m, J = 13.6 Hz,
cyclohex); δ 13C NMR (CDCl3): 168.7 (–O–CO–), 168.1 (–CO–
NH–), 155.4 (Ar′–C1), 154.4 (Ar–C1), 130.6 (Ar′–C2,6), 130.3
(Ar–C2,5), 129.2 (Ar′–C3,5), 129.0 (Ar–C3,5), 124.8 (Ar′–C4),
124.4 (Ar–C4), 72.7 ((CH2)2 > CH–O–), 70.5 (–CH2–CO–NH–),
20.0
589
in MeOH). CAS 1046053-08-0.
4.1.1.10. R-(−)-2-(2-(2,6-Dimethylphenoxy)acetamido)-
propanamide (10). (60%); mp 177–178; C13H18N2O3; δ H NMR
1
(CDCl3): 7.46 (1H, d, J = 6.4 Hz, –NH–CH–), 6.93–7.06 (3H, m,
Ar–H), 6.22–6.36 (1H, m, –NHH), 5.51–5.62 (1H, m, –NHH),
4.67 (1H, m, >CH–), 4.31 (2H, s, –O–CH2–CO–), 2.26 (6H, s,
Ar–(CH3)2), 1.51 (3H, d, J = 7.0 Hz, >CH–CH3); δ 13C NMR
(CDCl3): 173.7 (–CO–NH2), 169.0 (–CO–NH–), 154.3 (Ar–C1),
130.4 (Ar–C2,6), 129.2 (Ar–C3,5), 124.8 (Ar–C4), 70.3 (–CH2–
CO–), 47.9 (>CH–), 18.0 (>CH–CH3), 16.3 (Ar–(CH3)2); LCMS,
69.3 (–O–CO–CH2–), 46.7 (–NH–CH–(CH2)2<), 30.5 ((CH2)2
>
m/z: 251.14 (M+ + 1, 98.60%); HPLC, RT (B) = 2.92 min,
CH–O–),
29.9
(–NH–CH–(CH2)2<),
16.3
(Ar–(CH3)2,
20.0
589
100%; [α]
= −2.6710° (2% concentration in MeOH).
Ar′–(CH3)2); LCMS, m/z: 440.22 (M+ + 1, 100.00%); HPLC, RT
(B) = 14.21 min, 99.26%.
4.1.1.11. R,S-2-(2-(2,6-Dimethylphenoxy)acetamido)-N-
methylpropanamide (11). (55%); mp 129–131; C14H20N2O3; δ
1H NMR (DMSO-d6): 8.02 (1H, d, J = 8.5 Hz, NH), 7.95 (1H, q,
J = 4.6 Hz, –NH–CH3), 6.98–7.07 (2H, m, Ar–H3,5), 6.89–6.98
(1H, m, Ar–H4), 4.38 (1H, dq, J = 7.6 Hz; 4.7 Hz, >CH–), 4.27
(1H, d, J = 14.3 Hz, Ar–O–CHH–), 4.21 (1H, d, J = 14.3 Hz, Ar–
O–CHH–), 2.62 (3H, d, J = 4.7 Hz, –NH–CH3), 2.23 (6H, s,
Ar–(CH3)2), 1.28 (3H, d, J = 7.1 Hz, >CH–CH3); δ 13C NMR
(CDCl3): 167.6 (>CH–(CO)–NH–), 164.1 (–CH2–CO–), 149.7
(Ar–C1), 125.7 (Ar–C2,6), 124.4 (Ar–C3,5), 120.0 (Ar–C4), 65.6
(–CH2–CO–), 43.6 (>CH–), 21.6 (–NH–CH3), 13.7 (Ar–(CH3)2),
11.6 (–CH–CH3); LCMS, m/z: 265.17 (M+ + 1, 97.24%); HPLC,
RT (A) = 5.53 min, 99.84%.
4.1.1.15. R,S-N-(1-Hydroxybutan-2-yl)-2-(mesityloxy)acetamide
(15). (58%); mp 87–88; C15H23NO3; IR (KBr) ν = 3413, 3330,
2965, 2923, 1651, 1536, 1212, 1146, 1085, 1054, 986, 856, 661,
586, 574 cm−1; δ 1H NMR (CDCl3): 6.99–7.09 (1H, m, NH),
6.83 (2H, s, Ar–H), 4.20–4.35 (2H, m, Ar–O–CH2–CO–), 3.92–
4.03 (1H, m, >CH–), 3.75–3.81 (1H, m, –CHH–OH), 3.65–3.73
(1H, m, –CHH–OH), 2.24 (3H, s, Ar–CH3IJ4)), 2.22 (6H, s,
Ar–(CH3)2IJ2,6)), 1.54–1.77 (2H, m, –CH2–CH3), 0.97–1.07 (3H,
s, –CH2–CH3); δ 13C NMR (CDCl3): 169.7 (–CO–NH–), 152.1
(Ar–C1), 134.1 (Ar–C4), 129.9 (Ar–C3,5), 129.7 (Ar–C2,6), 70.5
(–CH2–CO–), 65.1 (–CH2–OH), 53.1 (–CH<), 24.2 (–CH2–CH3),
20.6 (Ar–CH3IJ4)), 16.2 (Ar–(CH3)2IJ2,6)), 10.5 (–CH2–CH3);
LCMS, m/z: 266.23 (M+ + 1, 97.42%); HPLC, RT (A) = 10.06
min, 99.55%. CAS 1156896-52-4.
4 . 1 . 1 . 1 2 .
R , S - 2 - ( 2 , 6 - D i m e t h y l p h e n o x y ) - N - ( 2 -
hydroxybutyl)acetamide (12). (60%); mp 96–97; C14H21NO3; IR
(KBr) ν = 3420, 3336, 2969, 2923, 2878, 1655, 1540, 1455,
1353, 1266, 1196, 1053, 791, 668, 570, 435, 418 cm−1; δ 1H
NMR (DMSO-d6): 7.95 (1H, t, J = 5.6 Hz, NH), 6.90–7.12 (3H,
m, Ar–H), 4.76 (1H, d, J = 5.2 Hz, OH), 4.21 (2H, s, –O–CH2–),
3.48–3.50 (1H, m, >CH–), 3.23–3.28 (1H, m, –NH–CHH–),
3.08–3.13 (1H, m, –NH–CHH–), 2.23 (6H, s, Ar–(CH3)2), 1.39–
1.48 (1H, m, >CH–CHH–). 1.26–1.35 (1H, m, >CH–CHH–),
0.88 (3H, t, J = 7.4 Hz, –CH2–CH3); δ 13C NMR (DMSO-d6) δ:
168.3 (>CO), 155.1 (Ar–C1), 130.8 (Ar–C2,6), 129.3
(Ar–C3,5), 124.7 (Ar–C4), 71.0 (>CH–), 70.7 (–CH2–CO–), 44.6
(–NH–CH2–), 27.8 (–CH2–CH3), 16.4 (Ar–(CH3)2), 10.3 (–CH2–
4. 1. 1. 16.
R, S-N-(2-Hydroxy-2-phenylethyl)-2-
(mesityloxy)acetamide (16). (60%); mp 121–123; C19H23NO3; IR
(KBr) ν = 3357, 2975, 2937, 2921, 2873, 1653, 1536, 1483,
1208, 1148, 1097, 1065, 1043, 854, 752, 702, 593, 573, 528,
1
412 cm−1; δ H NMR (CDCl3): 7.27–7.45 (5H, m, Ar′–H, NH),
6.82 (2H, s, Ar–H3,5), 4.96 (1H, m, OH), 4.26 (1H, s, Ar–O–
CHH–CO–), 4.25 (1H, s, Ar–O–CHH–CO–), 3.84 (1H, ddd, J =
13.8, 6.7, 3.5 Hz, 1H, >CH–), 3.47–3.61 (1H, m, –NH–CHH–
CH<), 2.92–3.31 (1H, m, –NH–CHH–CH<), 2.24 (3H, s,
Ar–CH3IJ4)), 2.17 (6H, s, Ar–(CH3)2IJ2,6)); δ 13C NMR (CDCl3):
170.1 (>CO), 152.0 (Ar–C1), 141.7 (Ar′C1), 134.1, (Ar–C4),
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130.0 (Ar–C2,6), 129.6 (Ar′–C2,6), 128.5 (Ar–C3,5), 127.9 (Ar′–
C4), 125.9 (Ar′–C3,5), 73.4 (>CH–OH), 70.3 (–Ar–O–CH2–),
46.7 (–NH–CH2–), 20.7 (Ar–CH3IJ4)), 16.1 (Ar–(CH3)2IJ2,6));
LCMS, m/z: 314.21 (M+ + 1, 100.00%); HPLC, RT (B) = 5.76
min, 99.10%.
CCDC 1832675–1832677 contain the supplementary crystallo-
graphic data.
4.3. Pharmacology
4.1.1.17. R,S-2-(2-(Mesityloxy)acetamido)butanamide (17).
(60%); mp 156–158; C15H22N2O3; IR (KBr) ν = 3368, 3198,
2963, 2930, 1656, 1536, 1484, 1424, 1210, 1147, 1062, 852,
Antiepileptic activity and neurological toxicity assays were
carried out by the Epilepsy Therapy Screening Program
(ETSP, previously known as the Anticonvulsant Screening
Program, ASP), Epilepsy Branch, National Institute of
Neurological and Communicative Disorders and Stroke,
National Institutes of Health in Rockville, MD, USA.20 For all
experiments male Carworth Farms no. 1 (CF1) mice (18–30 g)
and male Sprague Dawley rats (100–150 g for MES and 250–
300 g for hippocampal kindling) were used.
For compounds 1 and 14 testing was performed in a man-
ner consistent with a protocol approved by the Institutional
Animal Care and Use Committee at the University of Utah
(protocol 15-10007, PI: Wilcox), while for other compounds
with a protocol “Evaluation and Characterization of Novel
Therapeutics for the Treatment and Prevention of Epilepsy
and Countermeasures” (protocol 12-11011).
4.3.1. Maximal electroshock seizure (MES) test. The MES
were elicited by 60 Hz alternating current (50 mA in mice
and 150 mA in rats) delivered for 0.2 s via corneal electrodes,
primed with an electrolyte solution containing an anesthetic
agent (0.5% tetracaine HCl). Protection in the MES test was
defined as the abolition of the hindlimb tonic extension com-
ponent of the seizure.22
1
674, 650, 637, 434, 420, 407 cm−1; δ H NMR (DMSO-d6): 7.84
(1H, d, J = 8.2 Hz, –NH–CH<), 7.50 (1H, br s, –CO–NHH),
7.14 (1H, br s, –CO–NHH), 6.81 (2H, s, Ar–H3,5), 4.24–4.33
(1H, m, >CH–), 4.21 (1H, s, Ar–O–CHH–), 4.18 (1H, s, Ar–O–
CHH–), 2.17 (9H, s, Ar–(CH3)3), 1.57–1.82 (2H, m, –CH2–CH3),
0.84 (3H, t, J = 7.3 Hz, –CH2–CH3); δ 13C NMR (DMSO-d6):
173.4 (–CONH2), 167.9, (–CONH–), 153.0, (Ar–C1), 133.4 (Ar–
C4), 130.3 (Ar–C2,6), 129.8 (Ar–C3,5), 71.1 (–CH2–CO–), 53.3
(>CH–),
25.9
(–CH2–CH3),
20.7
(Ar–CH3IJ4)),
16.3
(Ar–(CH3)2IJ2,6)), 10.1 (–CH2–CH3); LCMS, m/z: 279.19 (M+ + 1,
100.00%); HPLC, RT (B) = 4.18 min, 99.74%.
4.2. Crystallography
Crystals suitable for X-ray diffraction analysis were obtained
by slow evaporation of the solvent at room temperature from
acetonitrile for 6 and 7 and from a mixture of acetone and
decane (1 : 1) for 12. Colourless crystals appeared within two
weeks.
Diffraction data for single crystals of 6 and 7 were col-
lected at 130 K using an Oxford Diffraction SuperNova four
circle diffractometer equipped with a Cu (1.54184 Å) Kα radi-
ation source, graphite monochromator and Oxford CryoJet
system for measurements at low temperature. Diffraction
data for 12 were collected at 100 K on a Bruker-Nonius Kappa
CCD four circle diffractometer equipped with a Mo (0.71069
Å) Kα radiation source. The structures were solved by direct
methods using SIR-97.35 All non-hydrogen atoms were re-
fined anisotropically using weighted full-matrix least-squares
on F2. Refinement and further calculations were carried out
using SHELXL.36 For molecular graphics ORTEP37 was used.
Compound 6. C13H19NO3, Mr = 237.29, crystal size = 0.56 ×
0.27 × 0.14 mm3, orthorhombic, space group P212121, a =
7.3307(4) Å, b = 20.5418(9) Å, c = 8.5970(4) Å, V = 1294.6(1)
Å3, Z = 4, T = 130(2) K, 19 025 reflections collected, 2497
unique reflections (Rint = 0.0581), R1 = 0.0952, wR2 = 0.2371
[I > 2σ(I)], Flack parameter −0.21IJ11).
Compound 7. C13H19NO3, Mr = 237.29, crystal size = 0.34 ×
0.22 × 0.10 mm3, orthorhombic, space group P212121, a =
7.3182(8) Å, b = 20.524(2) Å, c = 8.597(1) Å, V = 1291.2(3) Å3,
Z = 4, T = 130(2) K, 18 683 reflections collected, 2497 unique
reflections (Rint = 0.0741), R1 = 0.0913, wR2 = 0.2315 [I >
2σ(I)], Flack parameter 0.12(18).
Compound 12. C14H20NO3, Mr = 250.31, crystal size = 0.35
× 0.24 × 0.05 mm3, orthorhombic, space group Pna21, a =
21.431(1) Å, b = 8.483(2) Å, c = 7.516(3) Å, V = 1366.4(6) Å3,
Z = 4, T = 100(2) K, 6398 reflections collected, 2386 unique re-
flections (Rint = 0.0982), R1 = 0.0851, wR2 = 0.1979 [I > 2σ(I)].
4.3.2. Subcutaneous pentylenetetrazole (scMET) test. The
scMET test was conducted by administration of pentylene-
tetrazole (85 mg kg−1) dissolved in 0.9% NaCl solution to
mice into the posterior midline of its neck. A minimal time
of 30 min subsequent to subcutaneous administration of
pentylenetetrazole was used for seizure detection. Animals
not displaying fore and/or hind limb clonus, jaw chomping,
or vibrissae twitching (3 to 5 seconds) were classified as
protected.22
4.3.3. 6 Hz model test. Corneal stimulation at 6 Hz (44
mA) was delivered for 3 s to mice by a constant current de-
vice to induce a psychomotor seizure. The animals then
exhibited a ‘stunned’ posture associated with rearing, fore-
limb automatic movements and clonus, twitching of the vi-
brissae, and Straub tail. The experimental endpoint was pro-
tection against the seizure. The animal was considered to be
protected if it did not display this behavior.21
4.3.4. Hippocampal kindling test. A bipolar stimulating
electrode was stereotactically implanted in the ventral hippo-
campus (AP −3.6, ML 4.9, DV −5.0 from dura, incisor bar +5)
of adult male Sprague Dawley rats (250–300 g) under keta-
mine–xylazine anesthesia. Three anchor screws were attached
to the skull with dental acrylic cement. After the incision was
closed with sutures, the animal received a single dose of
Bicillin (60 000 units, i.m.) and was returned to his home
cage in the animal quarters. Animals were kindled according
to the procedure of Lothman and Williamson.38 After 1 week
animals were stimulated with suprathreshold trains for 200
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μA for 10 s, 50 Hz, every 30 min for 6 h on alternate days
until they were fully kindled. One week later, a single dose of
test substance (50 mg kg−1, i.p.) on the behavioral seizure
score and afterdischarge duration was assessed in a single
group of kindled rats respectively at 15 or 0, 15, 45, 75, 105
and 135 min after drug administration. Results obtained at
the various points were compared with the last control stimu-
lus delivered 15 min prior to drug administration. Thus, each
animal served as its own control. When a drug treatment was
observed to significantly lower seizure score and decrease
afterdischarge, a dose–response study was initiated.
4.3.5. Neurotoxicity. Neurological deficit was measured in
mice by means of the rotarod test. The mouse was placed on a
1 in. diameter knurled plastic rod rotating at 6 rpm. Neurotoxic-
ity was indicated by the inability of the animal to maintain
equilibrium on the rod for at least 1 min, which is defined as
falling off the rod at least three times. In rats, neurological defi-
cit was indicated by ataxia and a range of additional symptoms:
a circular or zigzag gait, abnormal body posture and spread of
the legs, tremors, hyperactivity, lack of exploratory behavior,
somnolence, stupor, catalepsy, loss of placing response, and
changes in muscle tone. A rat is considered impaired if it dis-
plays two or more of these abnormal behaviors.22
acidic protein (GFAP)-positive astrocytes); the rest (8%)
consisted of Tuj-1 positive neurons (neuronal class III
β-tubulin). However, in the next passages the population of
neurons gradually decreased leading to a pure population of
GFAP-positive astrocytes.
One day before the experiment, the cells were seeded at a
density of 10 000 cells per well in 96-well plates in a medium
containing a reduced amount of serum (1% FBS). The astro-
cytes were treated with compound 1 (dissolved in ddH2O) at
concentrations of from 10 to 200 μM for 24 hours. The con-
trol cultures were supplemented with the same amount of an
appropriate vehicle.
4.4.2. MTT assay. Cells were seeded at a density of 1 × 104
cells per well in 96 well plates. Following overnight culture,
the cells were then treated with increasing doses of com-
pound 6 and incubated for 24 h. Following cell exposure
to each drug for 24 h in 96-well plates, 10 μl MTT reagent
(Cayman) was added to each well and after 4 hours of incuba-
tion (37 °C, 5% CO2), the medium was aspirated and the
formazan produced in the cells appeared as dark crystals at
the bottom of the wells. Next, Crystal Dissolving Solution
(Cayman) was added to each well. Then the optical density
(OD) of each well was determined at 570 nm on a plate
reader (BIOTEK).
4.3.6. Quantification of effective dose and toxic dose. The
ED50 and TD50 were calculated for MES and rotarod tests
performed in rats, respectively, by means of a computer pro-
gram using probit analysis.22
4.4.3. NR assay. Cells were seeded at a density of 1 × 104
cells per well in 96-well plates. After 24 hours they were
treated with compound 6 at a concentration of 10–200 μM.
After 24 h the medium was removed from the cells and re-
placed with 100 μl of medium containing NR. The medium
containing NR (4 μg ml−1) was prepared by mixing NR solu-
tion in PBS (4 mg ml−1) with EMEM supplemented with FBS
(5%). After 3 h of incubation and microscopic examination of
the cells, the medium was removed and the cells were
washed with PBS.
Then, 150 μl of solubilization solution (50–96% ethanol,
49% distilled water and 1% glacial acetic acid) was added to
each well, and the plates were shaken on a microtiter plate
shaker for 10 minutes. Absorbance was determined at
540 nm (A540) and cytotoxicity was calculated as: cytotoxicity
(%) = (1 − (experiment A540/control A540)) × 100.
4.4. In vitro studies on astrocytes
4.4.1. Cell culture
Mouse astrocytes. An astrocyte cell line (ATCC, CRL-2541)
was used in the study. The cells were cultured under
standard conditions (37 °C, 5% CO 2) in DMEM medium
supplemented with 10% FBS and antibiotics.
Human astrocytes. The iPSC cells were kindly provided by
Damian Ryszawy, PhD (Department of Cell Biology, Faculty of
Biochemistry, Biophysics and Biotechnology, Jagiellonian
University, Cracow, Poland). The cells were cultured on 100
mm Petri dishes in Essential 8 Medium (ThermoFisher:
A1517001) supplemented with 1% pen–strep solution and
Y-27632 ROCK inhibitor (SCM075, MERCK Millipore) at a
concentration of 10 μM. The culture medium was changed
each day. Before culture the dishes were covered with
vitronectin at a concentration 0.5 μg cm−2 according to the
manufacturer's protocol (ThermoFisher: A14700). To obtain
the neural stem cells the iPSC cells were cultured for 7 days
in Gibco PSC Neural Induction Medium (ThermoFisher:
A1647801) supplemented with 1% pen–strep solution. The
medium was changed each day. We used astrocyte growth
medium (Sigma-Aldrich: 821-500) supplemented with 1%
pen–strep to differentiate the NSC cells. The medium was
changed each day for 17 days until the appropriate conflu-
ence of astrocyte culture has been achieved. The astrocyte
phenotype was then confirmed by GFAP staining. The purity
of the astrocyte population was about 92% (glial fibrillary
4.4.4. CV assay. Cells were seeded at a density of 1 × 104
cells per well in 96-well plates. After 24 hours they were
treated with compound 6 at a concentration of 10–200 μM. Af-
ter 72 hours, cells were fixed for 15 min in a solution of form-
aldehyde (3.7%), washed with PBS and subsequently stained
with 500 μl of 0.01% crystal violet solution for 10 minutes.
The dye that stained the cells on the plates was eluted by 500
μl CH3OH solution (25% V/V) of citric acid (1.33% m/V) and
sodium citrate (1.09% m/V), and the optical density of the
extracted dye was read with a spectrophotometer at 540 nm.
4.5. Metabolic stability
In vitro tests with liver microsomes isolated from male rats
(Sigma, Saint Louis, MO, USA) were designed according
to Słoczyńska et al.33 with minor modifications. The
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compounds, tested at a concentration of 20 μM, were
preincubated with rat liver microsomes (0.4 mg ml−1) in 100
mM potassium phosphate buffer (pH 7.4) for 15 min. The
proper experiment started with the addition of a regeneration
system consisting of 3.4 mM of NADP+, 7.7 mM glucose
6-phosphate and 0.6 U ml−1 glucose 6-phosphate dehydroge-
nase in potassium phosphate buffer (pH 7.4). Experiments
were carried out for 5 to 60 min at 37 °C in duplicate. Reac-
tions were stopped by adding cooled perchloric acid, followed
immediately by spiking samples with an internal standard.
Then, the samples were centrifuged at 9000 rpm for 10 min
at 4 °C. The supernatant was analyzed by LC-MS/MS analysis
(UPLC/MS, Waters Corporation, Milford, MA, USA). In vitro
half-life (t1/2) and intrinsic clearance (Clint) of the studied
compounds in liver microsomes were determined according
to literature procedures.39,40
substance solution (in DMSO), 0.05 ml of a mutagen (NaN3)
and 0.5 ml of
a phosphate buffer were mixed and
preincubated (37 °C, 30 min). 2 ml of top agar were added
and the mixture was poured onto the top of glucose minimal
agar plates. Revertant colonies per plate were counted after
the incubation (37 °C, 48 h). Each determination was made
in triplicate. The inhibition percentage of mutagenicity was
calculated using the following formula: [1 − ((S1 − S0)/(S2 −
S0))] × 100, where S0 is the number of spontaneous rever-
tants, S1 is the number of revertant colonies per plate in the
presence of both mutagen and test compound, and S2 is the
number of revertant colonies per plate in the presence of
mutagen only.42,44,45 The antimutagenic effect was defined as
weak or absent (inhibition up to 25%), moderate (25–40%
inhibition) or strong (40% or more inhibition).46,47
Conflicts of interest
4.6. Mutagenicity
There are no conflicts to declare.
4.6.1. Bacterial strains. Salmonella typhimurium strains
TA100 and TA1535 were kindly provided by Dr. T. Nohmi (Di-
vision of Genetics and Mutagenesis, National Institute of Hy- Acknowledgements
gienic Sciences, Tokyo, Japan).
The authors thank Jeff Jiang, Ph.D., James Stables, Ph.D.,
4.6.2. Chemicals and reagents. Magnesium sulfate, citric
acid monohydrate, potassium phosphate dibasic, sodium am-
monium phosphate, L-histidine, D-biotin, sodium phosphate
dibasic, monobasic sodium phosphate, magnesium chloride,
potassium chloride, sodium azide (NaN3), 2-aminoanthracene
(2AA), nicotinamide adenine dinucleotide phosphate, glucose-
6-phosphate and liver S9 fraction were purchased from Sigma-
Aldrich. D-Glucose and DMSO were supplied by Chempur,
nutrient broth no. 2 was obtained from Oxoid and agar was
purchased from Merck.
Prof. Steve White, Harvey Kupferberg, Ph.D., John Kehne, Ph.
D., Tracy Chen, Ph.D. and Shamsi Raeisi, Ph.D., from the
Nationa Institute of Neurological Disorders and Stroke (NIH,
Rockville, MD, USA) for providing the results of anticonvul-
sant evaluation, and Prof. Katarzyna Kieć-Kononowicz for co-
ordination of the cooperation between NINDS and the Faculty
of Pharmacy at Jagiellonian University Medical College. This
work was supported by the National Science Centre, Poland,
decision on grant no. DEC-2015/17/N/NZ7/00966 and statutory
funds K/DSC/003524, K/ZDS/007882 and K/ZDS/007884.
4.6.3. Mutagenicity testing. Mutagenicity was assessed
using the plate incorporation method with and without meta-
bolic activation. 100 μL of an overnight grown Salmonella
(TA100 or TA1535) culture, 50 μL of a test compound solution
(in DMSO), and 500 μL of a buffer solution (or S9 mix) were
mixed and preincubated (37 °C, 30 min). Next, 2 ml of top
agar were added to the tubes. Positive control plates
(containing NaN3 in the assay without metabolic activation
and 2AA in the assay with metabolic activation) and negative
control plates (vehicle) were performed alongside. The
contents of the tubes were mixed and poured onto the top of
glucose minimal agar plates and allowed to solidify. The
plates were incubated (37 °C, 48 h), and revertants growing
per plate in triplicate experiments were recorded.41,42 The
mutagenicity index (MI), the ratio between the number of
revertants per plate with a test compound and the number of
revertants observed in a negative control, was calculated.
Mutagenic activity results may be considered as positive
when at least a two-fold increase in the number of revertant
colonies (MI ≥2) is observed with at least one concentration
and a dose–response relationship is observed.42–44
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